1. Technical Field
The present disclosure relates to systems and methods for obtaining protein(s). More particularly, the present disclosure relates to systems and methods for obtaining protein(s) from a protein mixture, e.g., a binary protein mixture, by utilizing membranes. The membranes may be advantageously aligned in series. According to the disclosed systems and methods, ultrafiltration may be carried out on at least two proteins of substantially the same molecular weight simultaneously. In exemplary embodiments of the present disclosure, flat regenerated cellulose membranes and/or polyethersulfone membranes that possess substantially the same molecular weight cutoff (MWCO) are stacked together in a desired number to effectuate ultrafiltration, the efficacy of such stacking being further assisted by optimization of the feed conditions, membrane selection and/or membrane charge conditions to enhance/maximize selectivity.
2. Background of Related Art
Downstream protein purification from a fermentation broth is complicated by the presence of a large number of impurities, pyrogens, and viruses. Because a completely purified bioproduct is generally desired, many purification processes have been investigated, including chromatography, membrane adsorption or membrane chromatography, and ultrafiltration [Harrison R, Protein purification process engineering, New York: Marcel Dekker, 1994]. These techniques are successfully used in industry to purify biomolecules; however, the development of techniques that increase the selectivity and reduce the cost are highly desirable, making them the current focus of research.
Chromatographic processes achieve very high selectivities based on solute interaction with specific beads [Scopes R K., Protein purification principles and practice, 3rd Ed., New York: Springer Verlag, 1994]. In this process, separation is limited by diffusion in and out of the resin. Recent development of gigaporous particles facilitating convective flow through the particles is expected to mitigate this problem [Pfeiffer J F, Chen J C, Hsu J T, Permeability of gigaporous particles. AIChE J 42:932-939, 1996]. However, the buffer volume employed is very high. Scaleup is problematic and column chromatography is costly. In membrane adsorption processes, ligands are grafted on to the surface of pores in microfiltration membranes; biomolecule adsorption is achieved during convection through the membrane pores [Thömmes J, Kula M R, Membrane chromatography—an integrated concept in the downstream processing of proteins, Biotechnol Prog 11: 357-367, 1995]. This process is attractive because of the large pore size in microfiltration membranes, which allows much easier and convective access to the binding sites rather than diffusion in beads packed in chromatography columns. The ligand utilization is orders of magnitude higher. But it is not a steady state process. Overall capacity is low, therefore multiple cycles are needed.
The techniques mentioned above require specially designed adsorbents and unusual operating conditions. For example, membrane adsorption processes employ rapid cyclic procedures consisting of many cycles in which each cycle requires an adsorption, elution, and regeneration step. In addition, column chromatography is very costly. Membrane chromatographic processes have problems with dispersion in current device designs [Gebauer K H, Thommes J, Kula M R, Plasma protein fractionation with advanced membrane adsorbents, Biotechnol Bioeng 54:181-189, 1997], binding capacities comparable with beads have not been achieved [Sarfert F T, Etzel M R, Mass transfer limitations in protein separations using ion-exchange membranes, J Chromatogr A 764:3-20, 1997].
Ultrafiltration has traditionally been used for size-based separation of protein mixtures where the ratio of the protein molecular masses is at least around 7-10 [Cherkasov A N, Polotsky A E, The resolving power of ultrafiltration, J Membr Sci 110:79-82, 1996]. In order to achieve better purification of similarly sized biomolecules, significant research has taken place focusing on “fine tuning” the operating and physicochemical conditions to achieve higher selectivity [Saksena S, Zydney A L, Effect of solution pH and ionic strength on the separation of albumin from immunoglobulins (IgG) by selective ultrafiltration, Biotechnol Bioeng 43:960-968, 1994; Eijndhoven R H, Saksena S, Zydney A L, Protein fractionation using electrostatic interactions in membrane filtration, Biotechnol Bioeng 48 :406-414, 1995; Nystrom M, Aimar P, Luque S, Kulovaara M, Metsamuuronen S, Fractionation of Model Proteins using their Physiochemical Properties. Colloids and Surfaces A, Physiochemical and Engineering Aspects 138:185-205, 1998]. This allows the protein size differential to be exploited further due to the increased or decreased hydrodynamic radius that results from buffer conditions (i.e., ionic strength and pH). Some have employed these concepts along with a preliminarily determined optimal operating flux or transmembrane pressure drop to develop a technique called high-performance tangential flow filtration (HPTFF) [Van Reis R, Gadam S, Frautschy L N, Orlando S, Goodrich E M, Saksena S, Kuriyel R, Simpson C M, Pearl S, Zydney A L, High Performance Tangential Flow Filtration, Biotechnol Bioeng 56:71-82, 1997]. These HPTFF units can also be used in series to improve separation.
Others have examined theoretically an analogy between multistage ultrafiltration and size-exclusion chromatography [Prazeres D M F, A theoretical analogy between multistage ultrafiltration and size-exclusion chromatography, Chem Eng Sci 52:953-960, 1997]. A large stack of membranes was analyzed and compared to column chromatography for fractionation of solutes according to their size. It was suggested that such a multistage ultrafiltration-based chromatographic process behaves in an opposite fashion when compared to size-exclusion chromatography by eluting solutes in increasing order of their size. In this analysis, all of the solutes pass through the column at different rates depending on their size. No experimental data has been provided. The number of ultrafiltration membranes to be used in a stack could also number as high as 2500.
Sequentially-staged ultrafiltration membrane processes have been studied to separate aquatic humic substances [Burba P, Aster B, Nifant'eva T, Shkivnev V, Spivakov, B, Membrane filtration studies of aquatic humic substances and their metal species: a concise overview Part 1. Analytical fractionation by means of sequential-stage ultrafiltration, Talanta 45: 977-988, 1998]. Tangential flow-based filtration takes place through ultrafiltration membranes that are placed in series in different compartments with decreasing molecular weight cut offs (MWCOs) and allowed to collect in a reservoir after each stage. The flow rate across each membrane is controlled by a multi-channel pump.
Ultrafiltration, with optimized conditions, only improves the selectivities, not always resulting in a pure product. HPTFF requires extensive system optimization. Sequentially-staged ultrafiltration requires an extraordinarily large amount of equipment due to the multiple stages and pumps and has not received significant interest. This is because conventional multistage ultrafiltration is grossly inefficient in fractionating/purifying proteins having molecular weight ratios less than five [Ghosh, R, Novel cascade ultrafiltration configuration for continuous high-Resolution protein-protein fractionation: a simulation study, J Membr Sci 226:85-99, 2003]. Novel cascade configurations in separate devices with individual pumps have therefore been investigated to achieve protein purification by Ghosh [2003], who numerically illustrates such a three-stage process for protein fractionation using two proteins whose apparent sieving coefficients are 0.5 (preferentially transmitted) and 0.01 (preferentially retained).
In general, the advantages of membrane devices over chromatographic systems are lower capital cost and steady-state operation, which allow efficient transfer to large-scale operation. Ultrafiltration has become the preferred method of choice for protein concentration and buffer exchange, replacing size-exclusion chromatography [Kurnik R T, Yu A W, Blank G S, Burton A R, Smith D, Athalye A M, Van Reis R, Buffer exchange using size exclusion chromatography, countercurrent dialysis, and tangential flow filtration: models, development, and industrial application, Biotechnol Bioeng 45:149-157, 1995]. Changing the physicochemical environment as well as chemical modification of membranes have been extensively studied, and allow high-resolution protein separations [Saksena S, Zydney A L., Effect of solution pH and ionic strength on the separation of albumin from immunoglobulins (IgG) by selective ultrafiltration, Biotechnol Bioeng 43:960-968, 1994; Eijndhoven R H, Saksena S, Zydney A L., Protein fractionation using electrostatic interactions in membrane filtration, Biotechnol Bioeng 48 :406-414, 1995; Nystrom M, Aimar P, Luque S, Kulovaara M, Metsamuuronen S., Fractionation of Model Proteins using their Physiochemical Properties. Colloids and Surfaces A, Physiochemical and Engineering Aspects 138:185-205, 1998]. Laboratory as well as large-scale UF devices employ a simple flat membrane conventionally in a single stage [Kulkarni S S, Funk E W, Li N N, Ultrafiltration, in Ho W S and Sirkar K K editors, Membrane Handbook, Boston: Kluwer Academic Publishers, pp 393-453, 2001]. But by using a cascade operation in one stage, a single-staged optimized separation can be exploited to achieve very high selectivities that are characteristic of multiple stages that, until now, were only achievable using conventional column chromatographic methods. One device that yields a completely purified biomolecule by completely rejecting the unwanted species is highly desirable.
Typical membrane cascade operations are performed by transferring the permeate from one membrane device into a second device as the feed. In this research, multiple flat membranes are sandwiched together and housed in one device [Kulkarni S S, Funk E W, Li N N, Ultrafiltration, in Ho W S and Sirkar K K editors, Membrane Handbook, Boston: Kluwer Academic Publishers, pp 393-453, 2001]. The permeate from the first membrane is the feed for the second membrane, and the permeate from the second membrane is the feed for the third membrane, etc. Therefore, the rejection of one protein through one membrane will be substantially increased with each additional membrane eventually resulting in, on an overall basis, essentially complete rejection of one species. By using a membrane stack, e.g., a stack of 3, 4, or 5 membranes, it is possible to achieve an essentially completely pure product. We may also compensate for the solvent flux loss with each added membrane while still maintaining the effectiveness of the design by raising the feed solution pressure. However, the volume of the feed solution being imposed on each membrane, except for the first one, is very small.
Limited studies have been made where two asymmetric Omega 30K and 50K MWCO membranes were used in a sandwich, either with their support substructures together (i.e., the skin layers on the two outer surfaces) or with the skin layers together (i.e., with the porous substructures at the upstream and downstream surfaces) [Boyd R F, Zydney A L, Sieving characteristics of multilayer ultrafiltration membranes, J Membr Sci 131:155-165, 1997].
Despite efforts to date, a need remains for enhanced separation systems and methods, particularly separation systems and methods that are effective to separate proteins from protein mixtures in a cost effective and efficacious manner. Moreover, separation systems and methods are needed that can be effectively employed in protein systems wherein the ratio of the protein molecular mass is less than about 7 to about 10. These and other objectives are advantageously satisfied according to the disclosed systems and methods, as described herein.